In the field of genetic engineering, precise controlling gene expression provides a valuable tool for studying and manipulating development and other physiological processes. Gene expression is a complex biological progress in which many specific protein-protein interactions are involved. The first step is the transcription of DNA into RNA, which needs the delivery of transcription factors to the promoter of gene. Transcription factor, also known as a sequence-specific DNA binding protein, is a kind of protein or protein complex which is capable of binding specific DNA sequence of promoter to initiate or prevent the transcription of downstream gene into RNA by recognizing/binding to the promoter. There are two kinds of transcription factors, termed as transcriptional activator and transcriptional repressor. In general, a transcription factor contains at least a DNA binding domain which can recruit cofactor(s), also named as co-activators, to bind proper locus of the promoter together so as to initiate the gene transcription. Besides, a transcription factor also contains a transcriptional activation domain or a transcriptional repression domain; the transcriptional activation domain may be in a certain distance from the DNA binding domain. Conventional transgenic method for heterologous gene expression usually uses universal promoter or cell type-specific promoter. The construct with a target gene and a corresponding promoter is transfected into host cells or integrated into the genome, and the target gene is transcribed and further translated into the corresponding protein in specific cell type when binding the transcription factor to the promoter.
An alternative way to regulate heterologous gene expression in host cells is using inducible promoters, which includes two categories: 1) Chemical substance inducible promoter and gene expression systems, and 2) physical methods inducible promoter and gene expression systems. Chemical substance inducers include small molecule drugs, a typical example is antibiotics such as tetracycline [Gossen, M. and H. Bujard, Proc Natl Acad Sci USA, 1992, 89 (12): 5547-5551—Gossen, M., etc., Science, 1995, 268 (5218): 1766-1769], and streptomycin [M. etc., Nat Biotechnology, 2000, 18 (11): 1203-1208]; hormone [Wang, Y, Proc Natl Acad Sci USA, 1994. 91 (17): 8180-8184] and its analogues and other inducers such as acetaldehyde [Weber, W., etc. NAT Biotechnol, 2004, 22 (11): 1440-1444]. Physical methods inducible promoters and gene expression system include ultraviolet (UV)-regulated “cage” (Caged) technology [Keyes, W M and A A Mills, Trends Biotechnology, 2003, 21 (2): 53-55]; and far-infrared light controlled heat shock effect mediated gene expression system [Kamei, Y., etc., Nat Methods, 2009, 6 (1): 79-81].
Although many of those methods have been widely used, there exist some potential problems, such as (1) pleiotropic effect of the inducers may interfere with endogenous gene expression, which may lead to a complicated result. For example, in heavy metal ions-induced gene expression systems, heavy metal ions are not only able to induce target gene expression but also cause other heavy metal ion-induced endogenous gene expression; (2) some inducers have potential toxicity which can affect the functions of other genes, e.g., the cytotoxicity of heavy metal ions impedes their use in animals or humans; (3) Many promoter systems have a high leakage expression in the absence of inducers, thus the gene expression cannot be in a completely off state and the ratio of gene expression level before and after adding the inducers (also termed as induction ratio) is low, such as hormone-induced gene expression system [Wang, Y., and so on. Proc Natl Acad Sci USA, 1994, 91 (17): 8180-8184]. Those promoter systems are not suitable for expressing toxic genes or genes for that low expression level can cause significant biological effects; (4) Some chemical-induced gene expression systems consist of two or more proteins, such as the gene expression system based on FKBP-FRAP, and they have lower transfection efficiency than the single transcription factor [Rivem, V M, etc., Nat Med, 1996 2 (9): 1028-1032]. (5) Chemicals can temporally but not spatially regulate gene expression in specific cells and tissues. (6) Most physical methods have a strong toxicity to cells, e.g., the UV-induced cage technology may cause irreversible damage to cells, the inducible expression system based on far-infrared laser-controlled heat shock may activate endogenous gene expression, and the device is complex and expensive [Kamei, Y., etc., Nat Methods, 2009, 6 (1): 79-81].
However, light is a non-toxic inducer, which can be spatiotemporally controlled. One feasible way is introducing heterogenous light-regulated proteins (also known as photosensitive protein) into eukaryotic cells and reconstructing them to be light-regulated transcription factors which can regulate gene expression via light irradiation. These proteins are expected not to interfere with the physiological processes of eukaryotic cells, and they would not cause pleiotropic effects or non-specific effects in eukaryotic cells. However, studies on light-regulated transcription factors have been rarely reported. Shimizu-Sato et al. reported a light-switchable gene expression system in yeast cells [Shimizu-Sato S. et al, NAT Biotechnology, 2002 20 (10): 1041-1044]. U.S. Pat. No. 6,858,429 described using genetic engineering techniques to combine plant protein phytochrome (abbreviated as Phy) and Phytochrome interacting factor 3 (abbreviated as PIF3) with yeast Gal4 DNA binding domain and Gal4 transactivation domain of the yeast two-hybridization system to obtain fusion proteins Gal4-Phy and PIF3-GAD, respectively. Gal4-Phy interacts with PIF3-GAD and recruits PIF3-GAD to the target promoter to initiate gene transcription by red light illumination; while far-red light irradiation causes dissociation of the conjugate of Gal4-Phy and PIF3-GAD thus the AD domain of Gal4 cannot bind to the promoter and the transcription of target gene is terminated. This light-induced promoter system is reversible and has a high expression lever. However, the interaction of phytochrome Phy and PIF3 needs the existence of phycocyanobilin, the chromophore of PIF3, which needs to be added exogenously into yeast and mammalian cells since it does not exist in these cells. In addition, this system is based on yeast two-hybridization system in which the transcription factor consists of two proteins, and the resulting big construct is difficult to be introduced into host cells, thus limiting the wide usage of this system.
There are some other known photosensitive proteins: the photosensitive proteins using flavin as the chromophore (also called flavin-containing protein family blue light receptor), which can be divided into three groups: First is photoreceptors with light-oxygen-voltage (LOV) domain, such as phytochrome; the second is photolyase-like cryptochromes; the third is blue light using FAD (BLUF) family that is found in recent years. Phytochrome is the most common photoreceptor containing LOV domain, such as phototropin 1, white collar-1 (WC-1), white collar-2 (WC-2), photoactive yellow protein (PYP), Phy3, VVD, etc. Phytochrome is usually a membrane-coupled kinase which can autophosphorylate and alter its activity to regulate specific physiological processes upon blue light exposure. Most phytochromes have Serine/Threonine kinase domain at the C-terminal and two LOV domains with flavin at the N-terminal. With the illumination of blue light, the LOV domain and flavin bind covalently to form a cysteinyl-flavin adduct which can cause the conformation change of flavin-binding pocket and then enable the kinase domain at the C-terminal to alter the kinase activity. This process is reversible. In addition, LOV2 domain is more sensitive than LOV1. Based on the interaction of Arabidopsis FKF1 (flavin-binding, kelch repeat, f box 1) and GI (GIGANTEA) protein upon blue light irradiation, Masayuki Yazaw et al. fused FKF1 and GI to DNA binding domain of Gal4 and the transactivation domain AD of herpes simplex virus VP16 to form transcription factors Gal4-FKF1 and VP16-GI, respectively [Yazawa, M., et al., Nat Biotechnology, 2009. 27(10):941-945]. Upon blue light illumination, VP16-GI can interact with Gal4-FKF1 (specifically, the interaction happens between FKF1 and GI) which has bound to the promoter region, and initiate the transcription of target gene. The drawbacks of this system are that the large constructs containing FKF1 or GI gene are difficult to be transfected into cells, and the induction ratio is very low (the highest one is only 5-fold). Cryptochromes from Arabidopsis thaliana are the first separated blue light photosensitive plant proteins, of which some have been well studied, such as cryptochrome1 (CRY1), cryptochrome 2 (CRY2), phytochrome A (phyA) and phytochrome B (phyB). Based on the interaction of Arabidopsis CRY2 and CIB1 (CRY-interacting bHLH1) protein upon blue light illumination, researchers fused CRY2 and CIB1 to Gal4 DNA binding domain and Gal4 transactivation domain of the yeast two hybridization system to construct transcription factors Gal4-CRY2 and CIB1-GAD, respectively. CIB1-GAD interacts with Gal4-CRY2 which has bound to the promoter, and initiate the expression of target gene [Kennedy, M J. et al. Nat Methods 2010.7 (12):973-975]. Although it is unnecessary for this system to add an exogenous chromophore, it is still difficult to manipulate since the system contains two fusion proteins on the basis of the two-hybridization system. Furthermore, there is some leaky expression in the absence of light. All these drawbacks limit the wide application of this system. Difference between blue light photoreceptor proteins with BLUF domain and photo-receptor proteins with LOV domain is that no adduct is generated between BLUF and flavin after light irradiation, but it will lead to 10 nm red-shift absorbance due to the conformation change of chromophore. The most well studied BLUF domain containing photoreceptor is AppA, which is a repressor of anti-transcription from Rhodobacter sphaeroides. AppA and transcription factor PpsR combine to form AppA-PpsR2 complex and enable PpsR not to bind with DNA in darkness; bright blue light irradiation may enable AppA to dissociate from the complex, and the released PpsR forms a tetramer and bind to a specific DNA sequence to repress the gene transcription.
In previous studies, hormone receptors or receptor mutants can be used to regulate gene functions and activities of transcription factors. For example, in Cre/LoxP system, we can reconstruct Cre recombinase to regulate its nucleus localization by fusing ER, PR or GR to it; thus the reconstructed Cre recombinase can play its function roles in the nucleus with the existence of corresponding ligand. Fusing a hormone receptor to a transcription factor can enable the transcription factor to function under the regulation of hormone, for example, a hormone-tetracycline co-regulated gene expression system in which tetracycline-regulated transcription factor is fused with EcR or GR, thus the gene expression is regulated by both the hormone and tetracycline.
As described above, the most widely used gene expression systems today utilize chemical substances as the inducers, which have reasonable desirable induction performance, low leakage expression and high expression levels. However, many of gene expression systems have side-effect and potential toxicity due to their pleiotropic effect. Besides, the chemical inducers cannot precisely control gene expression at high spatial resolution. Up to now, there are only a few of gene expression systems controlled by physical methods although they have the capability of spatial regulating gene expression in specific cells and tissues, their toxicity to host cells may cause irreversible damage or hard manipulation. Few photosensitive protein based gene expression systems have been developed, but the poor induction capacity, the requirement for exogenous chemicals, the difficulty for the transcription factor containing more than one protein to be introduced into host cells may limit their wide application. The applicants consider that a more excellent gene expression system can be created using a novel method to overcome the shortcomings of previous studies, and it can be widely used in biomedical sciences and technology researches. After painstaking studies, the applicant has invented a novel light-controllable gene expression system, which consists of two parts: a recombinant light-switchable transcription factor and a target transcription unit. It has excellent capacity to control gene expression and it can spatiotemporally regulate gene expression. Furthermore, in order to satisfy more complex synthetic biology research, we have modified the recombinant light-switchable transcription factor into a recombinant light-hormone dual regulated transcription factor which can regulate gene expression by both light and hormone.
Accordingly, the first object of the present invention is to provide a novel light-controllable gene expression system.
A second object of the present invention is to provide a light and hormone dual-regulated gene expression system.
A third object of the present invention is to provide a eukaryotic expression vector containing said light-controllable gene expression system.
A fourth object of the present invention is to provide a method of the regulation of gene expression by said light-controllable gene expression system in the host cell.
A fifth object of the present invention is to provide a kit containing components of the light controllable gene expression system.
A sixth object of the present invention is to provide a gene therapy method using the light-controllable gene expression system.